Lessons learned: establishing a CLIA-equivalent laboratory for targeted mass spectrometry assays - navigating the transition from research to clinical practice.

Mass spectrometry (MS) assays offer exceptional capabilities in high multiplexity, specificity, and throughput. As proteomics technologies continue advancements to identify new disease biomarkers, transition of these innovations from research settings to clinical applications becomes imperative. To meet the rigorous regulatory standards of clinical laboratories, development of a clinical protein MS assay necessitates adherence to stringent criteria. To illustrate the process, this project focused on using thyroglobulin (Tg) as a biomarker and an immuno-multiple reaction monitoring (iMRM) MS-based assay as a model for establishing a Clinical Laboratory Improvement Amendments (CLIA) compliant laboratory within the Centers of Genomic and Precision Medicine, National Taiwan University. The chosen example also illustrates the clinical utility of MS assays to complement conventional immunoassay-based methods, particularly in cases where the presence of autoantibodies in 10-30% of patients hinders accuracy. The laboratory design entails a comprehensive coordination in spatial layout, workflow organization, equipment selection, ventilation systems, plumbing, electrical infrastructure, documentation procedures, and communication protocols. Practical aspects of the transformation process, including preparing laboratory facilities, testing environments, instrument validation, assay development and validation, quality management, sample testing, and personnel competency, are discussed. Finally, concordant results in proficiency testing demonstrate the harmonization with the University of Washington Medical Center and the quality assurance of the CLIA-equivalent Tg-iMRM MS assay established in Taiwan. The realization of this model protein MS assay in Taiwan highlights the feasibility of international joint development and provides a detailed reference map to expedite the implementation of more MS-based protein assays in clinical laboratories for patient care.

ACK1 upregulated the proliferation of head and neck squamous cell carcinoma cells by promoting p27 phosphorylation and degradation.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy with a worldwide distribution. Although intensive studies have been made, the underlying oncogenic mechanism of HNSCC requires further investigation. In this study, we examined the oncogenic role of activated Cdc42-associated kinase 1 (ACK1), an oncogenic tyrosine kinase, in regulating the proliferation of HNSCC cells and its underlying molecular mechanism. Results from immunohistochemical studies revealed that ACK1 was highly expressed in HNSCC tumors, with 77% (77/100) of tumors showing a high ACK1 immunoreactivity compared to 40% (8/20) of normal mucosa. Knockdown of ACK1 expression in HNSCC cells resulted in elevated p27 expression, reduced cell proliferation, and G1-phase cell cycle arrest. Rescue of ACK1 expression in the ACK1-knockdown cells suppressed p27 expression and restored cell proliferation. Compared to ACK1-knockdown cells, ACK1-rescued cells exhibited a restored p27 expression after MG132 treatment and showed an elevated level of ubiquitinated p27. Our data further showed that knockdown of ubiquitin ligase Skp2 resulted in elevated p27 expression. Importantly, the expression of p27(WT), p27(Y74F), or p27(Y89F) in ACK1-overexpressed 293T cells or ACK1-rescued SAS cells showed higher levels of tyrosyl-phosphorylated p27 and interaction with ACK1 or Skp2. However, the expression of p27(Y88F) mutant exhibited a relatively low phosphorylation level and barely bound with ACK1 or Skp2, showing a basal interaction as the control cells. These results suggested that ACK1 is highly expressed in HNSCC tumors and functions to promote cell proliferation by the phosphorylation and degradation of p27 in the Skp2-mediated mechanism.

Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.